1. Technical Field
The present invention relates to the field of molecular biology and more specifically towards gene targeting by homologous recombination.
2. Background Art
Gene targeting by homologous recombination is one of the most important tools of modern molecular biology. Gene targeting can generate targeted “knockout” or “knock-in” mutations in a variety of model organisms such as mice, which facilitates the development of mouse models of human genetic disorders. Gene targeting can also be utilized to correct inherited diseases in humans through gene therapy.
Generally, homologous recombination repairs double strand breaks made in meiotic prophase or during the S and G2 phases of the cell cycle, whereas non-homologous end joining repairs double strand breaks generated in the absence of a sister chromatid (Goedecke et al., 1999; Pâques and Haber, 1999; Takata et al., 1998). However, this division of labor is not absolute. Several lines of evidence indicate that these pathways can act contemporaneously or even in concert. For example, homologous recombination can occur during G1 (Fabre, 1978), non-homologous end joining and homologous recombination compete for repair of transfected linear DNA molecules (Roth and Wilson, 1985), disabling one pathway increases the activity of the other (Pierce et al., 2001), and the two mechanisms can act in a coupled fashion to repair a double strand breaks (Richardson and Jasin, 2000). The precise manner in which a particular repair pathway is selected to repair a given DSB, or even whether such a selection is made, has remained unknown. Yet, the choice of repair mechanism can serve as an important control point in various types of reactions involving double strand break intermediates. One model posits that the choice of pathway is essentially stochastic, determined by whether the broken ends are bound by Ku or by, for example, Rad52 (leading to NHEJ or homologous recombination, respectively) (Goedecke et al., 1999; Van Dyck et al., 1999; Haber, 1999). Alternatively, it has been proposed that if the ends remain intact they are available for NHEJ, but 5′ strand resection can cause homologous recombination to occur (Frank-Vaillant and Marcand, 2002).
Recent scanning force microscopy studies indicate that Rad52 and Ku actually prefer different DNA substrates, and suggest that resection precedes Rad52 binding (Ristic et al.,2003). The identity of the “gatekeeper” molecule(s) that control this critical choice remains an open question. Adding to the puzzle is evidence suggesting that there can be other repair pathways, for example, the poorly characterized “alternative NHEJ” pathway that operates in the absence of classical NHEJ (i.e., Ku, XRCC4, DNA ligase IV, Artemis, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)) (reviewed in Ferguson and Alt, 2001; Roth, 2003).
There are indeed mechanisms to guide broken DNA ends to an appropriate pathway, which have been discovered in the context of a recombination system that introduces site-specific double strand breaks and depends upon repair by a particular pathway to rejoin the DNA ends:V(D)J recombination. As the DNA rearrangement process that assembles antigen receptor genes during lymphocyte differentiation, V(D)J recombination relies on classical NHEJ to join site-specific double strand breaks. It was recently discovered that the V(D)J recombinase (the RAG proteins) shepherds the double-strand breaks to the classical NHEJ pathway [Lee et al., Cell, 2004).
As shown in FIG. 1, recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce nicks at recombination signal sequences. Upon synapsis, the RAG proteins convert these nicks to double strand breaks, leaving four ends (two hairpinned coding ends and two blunt signal ends) (Roth, 2003). Whereas signal ends are blunt ends that can be directly joined, the covalently sealed hairpins must be opened and processed before the coding ends can be joined. End-processing and joining are carried out by the classical NHEJ factors (Roth, 2003) along with the help of the RAG proteins themselves, which maintain the ends in a post-cleavage complex in vitro (Agrawal and Schatz, 1997; Hiom and Gellert, 1998; Jones and Gellert, 2001) and in vivo (Qiu et al., 2001; Yarnall Schultz et al., 2001; Huye et al., 2002). In vivo studies of NHEJ mutants and mutant RAG proteins that have defects in joining demonstrate that the post-cleavage complex serves as a scaffold for the four ends to facilitate repair (Zhu et al., 1996; Qiu et al., 2001; Yarnall Schultz et al., 2001; Brandt and Roth, 2002; Huye et al., 2002).
Double strand breaks created during V(D)J recombination are deliberately shepherded by the RAG post-cleavage complex into a specific nonhomologous end joining pathway and the RAG post-cleavage complex prevents RAG-generated double-strand breaks from being repaired by alternative joining pathways. Mutations in RAG-1 disrupt the post-cleavage complex in a manner to allow double strand breaks to escape from the nonhomologous end-joining pathway and participate in homologous recombination.
As is well known in the art, current methods of gene targeting utilize a cell's endogenous homologous recombination machinery to integrate a gene. In prior studies, it was realized that linearizing the gene within the homology region greatly stimulates targeting by homologous recombination, and virtually all gene targeting protocols involve generating a double-strand break in the targeting vector (Vasquez, K M et al. Proc Nat Acad Sci. 98(15) 8403-8410). Double-strand breaks are common intermediates in a variety of genetic recombination processes. Double-strand breaks, however, are damaging toward chromosomal integrity because they cause aberrant chromosome rearrangements as well as deletion of essential genetic information. Therefore, targeted integrations are typically quite rare, occurring at a rate of approximately one event per 105 to 107 transfected cells (Sargent, R G and Wilson J H Curr. Res. Mol. Ther. 1: 584-692). Moreover, undesired and non-targeted events are always much more frequent than the desired homologous integration events. In somatic cells, nontargeted integrants are at least 1000-fold more frequent than targeted events (Sargent, R G and Wilson J H Curr. Res. Mol. Ther. 1 584-692). Although this ratio is more favorable in mouse embryonic stem cells, hundreds of integration events must still be searched to detect the desired targeted clone. As a result, a large amount of reagents, time, and energy must be expended to achieve the detection of the desired clone. Thus, even a modest (2-fold) increase in the ratio of targeted to non-targeted genetic modification events would be commercially useful.
The non-targeted integrations result from joining of the broken ends of the linearized targeting vector to endogenous chromosomal breaks by the non-homologous end-joining (NHEJ) pathway. In essence, these non-targeted integrations are necessary byproducts of linearizing the targeting vector to stimulate homologous recombination. In other words, placing a double-strand break in the targeted gene stimulates both the homologous recombination of the targeted gene and the undesired, non-targeted integrations. A modest increase in efficiency (around 5-fold) is gained through the use of positive and negative selection techniques; however, efficiency is still quite low. Thus, the identification of targeted clones is a significant hurdle in the standard practice of generating targeted ES cells, which are the precursors of “knockout” and “knock-in” mice, and is often prohibitively difficult for somatic cells.
Accordingly, there is a need for compositions, kits, and methods useful in stimulating homologous recombination and therefore useful in identifying targeted clones. More specifically, there is a need for improved gene targeting methods by homologous recombination wherein double-strand breaks are avoided. Further, there is a need for mutant or non-mutant proteins and compositions that stimulate homologous recombination.